Nearly all licensed vaccines protect through antibodies rather than cell-mediated immunity. A critical aspect of vaccine-induced serological protection is the duration of antibody titer post-boost. Initial T cell-B cell interactions can generate a population of short-lived, high-rate antibody secreting cells (the extrafollicular antibody response), which contributes to acute host defense but not to long-lived protection. The latter involves migration of antigen-activated T cells and the associated antigen-specific B cells into the B cell follicle where they set up the germinal center reaction. Here continued T-B interaction leads to somatic hypermutation and isotype class switching, producing antibodies of higher affinity and different effector class, while generating both memory B cells and also plasmablasts that can become long-lived plasma cells in the proper niche. While this general outline is well-established, the molecular signals that guide the engagement of T and B cells to generate a maximally productive response, the role of Tfh in determining the choice between memory B cells and long-lived plasma cells, and what determines how plasmablasts become long-lived plasma cells remain unclear. This project is a collaborative effort among laboratories with expertise in vaccine development, adjuvant function, and the cellular immune reactions that aims to examine how adjuvants affect each of these key steps in humoral immune responses and how variations in the quality and quantity of Tfh, stimuli for B cells, and niche space for plasma cells affects the peak titer and persistence of antibody responses post-vaccination. In FY15, progress has been made in moving from last years systematic testing of adjuvants with candidate antigens selected for a transmission-blocking malarial vaccine to the development of a model system that permits quantification and isolation of antigen-specific Tfh of relevance for conjugate vaccines and analysis of plasma cell numbers and quality. Distinct adjuvants produced substantially different peak post-boost levels of Pfs25-specific antibodies, with a role also found the carrier protein. Surprisingly and in contrast to data from NHP studies performed previously, the decline in antibody titer over nearly a year showed a similar slope for all conditions. This raises the important question of whether this is a species-related difference or if it reflects the fact that the mice are clean in comparison to the NHP, influencing the availability of bone marrow niche space for long-lived plasma cells. This issue is being addressed in mouse experiments with studies in conventionally reared animals and in those given strong infectious challenges to fill up the bone marrow plasma cell niche. These are long term studies that began in FY14 and have continued this FY. We have also initiated studies examining the role of inflammatory signals in mobilizing bone marrow resident plasma cells and allowing their replacement with newly generated plasmablasts derived from vaccination. Additional experiments are examining whether the physiological state of the plasmablasts also controls the duration of the serological response, with preliminary studies showing that we can detect differential rates of titer decline in mice given plasmablasts from different priming regimens. To complement these functional studies, we have developed new methods for long bone whole mount microscopy and for clearing of bone /bone marrow that allows detection of antigen specific and well as IgG producing plasma cells and the detailed characterization of the niche in which they reside in 3D. In prior work, we used a model system in which mice were immunized with a peptide epitope from Toxoplasma emulsified in Complete Freunds Adjuvant and then measured T cell responses using a specific Class II MHC tetramer at day 14 post-injection. Because neither this model antigen nor others in routine use for mouse experiments was directly relevant to the conjugate vaccines being examined for field use in transmission blocking studies, we have developed a new model that circumvents this limitation. Taking advantage of an algorithm developed in M. Jenkins laboratory for identification of peptide epitopes binding to the mouse MHC class II molecule I-Abb, we have identified several peptides from tetanus toxoid (TT) that are immunogenic in C57Bl/6 mice and that stimulate a recall response form animals immunized with the intact protein or with a Pfs25-TT conjugate. Using these peptides, tetramers were produced that detect specific Tfh in immunized animals and preliminary experiments indicate that adoptive transfer of these cells augments weak alum adjuvant-induced germinal center formation. This new model system will permit direct and quantitative analysis of Tfh formation via dendritic cell and antigen-specific B cell interactions, assessment of B cell activation by antigen and innate stimuli, examination of the outcome of Tfh-B cell interactions in germinal centers, and determination of the proportion of memory B cells vs. plasmablasts formed by this latter response. Of particular focus will be whether different adjuvants affect only Tfh number, Tfh quality, or both, based on use of the adoptive transfer model and quantification of transferred Tfh using these new TT tetramers. The data generated by these rodent experiments will be used to plan NHP studies focused on determining if the top candidate adjuvants produce the same types of responses in these primates at the macro (i.e., antibody titer) level and with respect to the cellular events involved, such as bone marrow niche regulation. A key aim of this project is the identification of vaccine formulations that will extend the duration of the antibody response against malaria vaccine candidates. Our prototype target malaria antigen is Pfs25, which is undergoing Phase 1 trials in humans as a Pichia-expressed recombinant protein conjugated to the carrier protein ExoProtein A (EPA) expressed in E. coli with a molar ratio of 3:1, and formulated with the commercially available adjuvant Alhydrogel: Pfs25-EPA/Alhydrogel. Pfs25-EPA/Alhydrogel has completed Phase 1 trials in malaria-naive volunteers in the US (dose-escalating trial) and malaria-experienced volunteers in Mali (dose-escalating; double-blinded; placebo-controlled trial). By ELISA, serum titers against the Pfs25 antigen increased with each boost up to 4 doses in both US and Malian populations, though mean Malian titers were only half those of US volunteers after the final dose. Importantly, the rate of decline of antibody against Pfs25 was significantly greater than that against EPA, suggesting that long-lived antibody-secreting cells may fail to develop against the target antigen. Highly functional antibodies (measured in Membrane Feeding Assays) that block parasite transmission to mosquitoes developed in many US and Malian vaccinees, and correlated with antibody titer; in addition, avidity assays showed that antibody avidity increased significantly with successive vaccine doses. The Pfs25-EPA/Alhydrogel product will be a benchmark against which we will compare novel Pfs25 products and formulations in our animal studies. Our animal studies of adjuvants to date support our plan to test Pfs25-based conjugate vaccines using alternative adjuvants in humans, and our initial focus is on the commercial product AS01 from GSK, with whom we have executed an agreement to conduct an additional preclinical study of Pfs25-EPA + Pfs230-EPA in AS01 prior to initiating toxicology studies and a subsequent human trial. As part of the ongoing collaboration in this project, we are now systematically measuring levels of plasmablasts and Tfh cells in animal and human studies, and ASC in bone marrow in animal studies, to relate to sustained antibody titers.